Stroke is the most prevalent cause of adult disability in the United States. Each year, more than 800,000 stroke patients acquire lifelong sensory, motor, and cognitive disability. There are currently no medical therapies to promote brain recovery after stroke. The surviving brain tissue, however, is capable of a limited degree of self-repair in the weeks to months after stroke, a process that can lead to functional improvement. Studies have identified that stroke triggers cortical reorganization and axonal sprouting in the surviving peri- infarct cortex. Initial studies indicate that new connections may form after stroke in an activity-dependent manner. The 2006 landmark EXCITE clinical trial found that stroke patients who engage in constraint-induced movement therapy, a behavioral paradigm for focused limb overuse, demonstrate significant and lasting motor improvements. This clinical finding and preclinical axonal sprouting studies suggest that activity-based neurorehabilitation may specifically promote the formation of new brain connections during recovery after stroke. However, the exact neuronal connections formed by activity-induced axonal sprouting have not been identified, and the molecular substrates that drive the formation of these repair circuits are unknown. We hypothesize that a specific ensemble of genes drives circuit rewiring during post-stroke limb overuse, a clinically relevant rehabilitative therapy that interfaces injury and activity-dependent molecular processes. This project will aim to understand activity-dependent mechanisms of circuit reorganization in a mouse model of cortical stroke. We have developed a novel limb-overuse paradigm analogous to human constraint- induced movement therapy. The studies in this proposal will first quantitatively map the specific brain circuits that rewire during limb overuse after stroke using fluorescent neuronal tracers. Then, these very circuits will be isolated by FACS (Fluorescence-Activated Cell Sorting) for RNA deep-sequencing of the genes that are triggered during the activity-based behavioral paradigm. Candidate gene systems will be tested in an in-vitro neuronal assay and prioritized for in-vivo genetic modification within specific stroke repair circuits. The results of these studis will generate molecular targets for enhancing adult neuroplasticity and functional recovery after stroke. This proposed research strategy outlines our circuits-to-molecules approach to take the principles in this field to the next level of understanding, a necessary step in their translation toward novel medical therapies for stroke recovery.